Materials and Methods for Treatment of Cancer

ABSTRACT

Glypican 5 is shown for the first time to have a role in proliferation of cancer cells, including tumours which do not show chromosomal amplification at 13q31. The use of glypican 5 (GPC5) antagonists and binding agents for the treatment of cancer, particularly rhabdomyosarcoma and breast cancer, is disclosed.

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer, and inparticular to the use of glypican 5 (GPC5) antagonists and bindingagents for the treatment of cancers.

BACKGROUND TO THE INVENTION

Amplification of genomic regions is frequently observed in human tumorsand is one mechanism leading to the upregulation of genes that maycritically affect cellular behaviour and drive tumour progression.Therefore, identifying the genes involved in amplification eventsrepresents a useful approach to increasing understanding of tumorigenicprocesses and may provide clinically useful markers.

Rhabdomyosarcomas (RMS) are the most common soft tissue sarcomas ofchildhood and account for around 5% of all childhood cancers. There aretwo main histological subtypes of RMS namely, alveolar (ARMS) andembryonal (ERMS). Both subtypes consist of cells that resemble and havemarkers for developing skeletal muscle. The alveolar subtype isgenerally associated with a poorer prognosis than ERMS and often has at(2;13)(q35;q14) or t(1;13)(p36;q14) translocation which fuses the PAX3or PAX7 genes, respectively, to FOXO1A (1-3). In addition to thesetranslocations a number of other aberrations have been defined includingregions of genomic amplification (4-10). There are a small butsignificant group of ERMS which demonstrate a poor response totreatment; the genetics of this group is not well defined.

Our previous work on RMS showed amplification of the 13q31-q32chromosomal region in around 20% of ARMS studied (4). In addition,samples from a number of other tumour types have been reported withamplification of the 13q31-32 region including other sarcomas(leiomyosarcomas (11), malignant fibrous histiocytomas (12), lymphomas(13), breast cancers (14), small cell lung carcinomas and variousneurological tumours (15-17)). Also, the widely available leukaemic cellline K562 has been shown to have amplification of the 13q31-32 region inaddition to the translocation associated with the BCR-ABL fusion gene(18). Recent work on a few lymphoma cell lines derived from differenttypes of lymphoma defined a minimum region of amplification at 13q31-32to an approximately 4 megabase region (13). This region contained theglypican 5 gene (GPC5) which was shown to be expressed and was suggestedas a possible target for the amplification event in lymphomas. Whetherthis gene plays a functional role in lymphomas and whether the same geneis involved in other tumour types with genomic aberrations in thisregion remains to be determined.

In a previous analysis of RMS samples we used a new approach to profileglobal changes in differential expression which targets chromosomescalled comparative expressed sequence hybridization (CESH) (19). Thedata from 45 cases was used in a study to examine the classificationpotential of these profiles (20). Here we compare chromosomal levelgenetic and expression data for the 13q31-32 region and suggest thatamplification is not the only mechanism leading to increased expressionof gene(s) from this region. In view of the frequent differentialexpression from the 13q31-32 region in addition to its amplification wehave sought to implicate gene(s) from this region in the development ofRMS. This could provide a target for therapeutic approaches to treatthese, and potentially other tumours.

SUMMARY OF THE INVENTION

As described above, amplification of 13q31 has been observed in alveolarRMS and a number of other cancer types. Yu et al. (13) have furthershown that the GPC5 gene is overexpressed in lymphoma cell lines havingan amplicon at 13q31-32, as compared to cell lines lacking thatamplicon. While those authors speculated that GPC5 might play a role inthe pathogenesis of lymphomas with amplification of 13q31-32, they didnot provide any evidence of this. Tumour cells are notoriouslygenetically unstable, being prone to acquiring genetic abnormalities,such as chromosomal amplifications, after transformation. It istherefore possible that the observed amplification was acquired aftertransformation, or alternatively is simply not involved in thetransformation process. Accordingly, there is no proof in the literatureto date that GPC5 has any role in normal or abnormal cell proliferation.

The present inventors have demonstrated that downregulation of GPC5expression in cells which overexpress it reduces the ability of thesecells to form colonies in vitro. The inventors have therefore shown forthe first time that GPC5 expression is directly linked to cellularproliferation, thus providing a novel therapeutic target.

Furthermore, the inventors have found that GPC5 is overexpressed intumours which do not show chromosomal amplification at 13q31.

The inventors have also found evidence to suggest that GPC5 may beregulated by the Wilms' Tumour (WT1) gene product. WT1 is a zinc fingertranscription factor which has been shown to be inappropriately and/orover expressed in leukaemias and a wide range of solid tumours includingprostate, breast and lung, as well as thyroid, testicular and ovariancarcinomas, melanoma and mesothelioma (reviewed in Reddy, J. C., andLicht, J. D. (1996) Biochim Biophys Acta 1287, 1-28; Scharnhorst, V.,van der Eb, A. J., and Jochemsen, A. G. (2001) Gene 273, 141-161). Thefact that GPC5 is associated with MYCN and WT1 which are genes of knownsignificance in tumours is consistent with GPC5 itself being ofimportance in tumorigenesis.

In tumour cells grown either in vitro or in vivo, downregulation of WT1results in the concomitant downregulation of GPC5. Conversely,upregulation of WT1 results in the upregulation of GPC5. Directtranscriptional regulation of GPC5 by WT1 is likely since GPC5 has twoWT1 consensus binding sites in its promoter region. Overexpression ofWT1 may therefore drive the overexpression of GPC5 in tumours which donot carry chromosomal 13q31-32 amplicons and GPC5 expression may bemodulated indirectly by targeting the expression of WT1. Downregulationof WT1 using antisense oligonucleotides results in inhibition of cellproliferation and induction of apoptosis (Algar, E. M., Khromykh, T.,Smith, S. I., Blackburn, D. M., Bryson, G. J., and Smith, P. J. (1996)Oncogene 12, 1005-1014). Inhibition of proliferation may be wholly orpartially mediated by downregulated GPC5 in some cell types.

Expression of high levels of WT1 is associated with poor prognosis inleukaemias and breast cancer. Evidence in the literature suggests thatWT1 may contribute to drug resistance mechanisms through interferencewith cell checkpoint control and apoptotic pathways. However definitiveevidence is lacking. The inventors have demonstrated upregulation of WT1in chemoresistant tumour cell lines treated with cytotoxic drugs, aphenomenon absent in sensitive cell lines. Associated upregulation ofGPC5 has also been demonstrated. Upregulation of GPC5 may mediate someof the effects of upregulated WT1 and contribute to chemoresistancemechanisms. Similarly, overexpression of GPC3 has recently beenimplicated in resistance to mitoxantrone and etoposide in a cell linemodel (Wichert et al. Oncogene 23: 945-955 2004). Inhibition of GPC5activity either by direct or indirect downregulation of expression, orby blocking its activity, may increase the potency of some classes ofcytotoxic drugs, particularly in cancers which inappropriately expressor overexpress WT1.

The inventors' work therefore suggests a number of ways in which GPC5may be targeted therapeutically. GPC5 is a cell surface molecule; agentscapable of binding to GPC5 may therefore be used to direct therapeuticagents to target cells. Additionally or alternatively, antagonists whichinhibit the expression or function of GPC5 at the cell surface can beused to inhibit cell proliferation directly. Furthermore, such GPC5antagonists may also be used to increase the sensitivity of target cellsto other chemotherapeutic agents, and so may be of significance intreating tumours that have become resistant to therapy.

Thus the therapeutic application of the inventors' findings extends farbeyond those few cancers carrying chromosomal 13q31 amplicons.

Thus, in a first aspect, the present invention provides a method ofinhibiting proliferation of a target cell, comprising contacting thecell with a GPC5 antagonist or a GPC5 binding agent.

In this aspect of the invention, a GPC5 binding agent is typically anagent capable of binding to GPC5 protein, that is to say to the GPC5core protein and/or its associated heparan sulphate chains. PreferredGPC5 binding agents are antibodies, although peptides and small moleculebinding agents may also be suitable.

The GPC5 binding agent may be used to direct a therapeutic agent capableof inhibiting proliferation of the target cell to the appropriate celltype. Thus the method may comprise the step of contacting the cell witha therapeutic agent.

The therapeutic agent may be part of, or associated with (covalently ornon-covalently bonded or otherwise linked to), the binding agent.Alternatively the binding agent may be used to label a target cell inorder that a suitable therapeutic agent can then be directed to the cellin preference to unlabelled cells, or activated in the vicinity of thecell. In such embodiments the therapeutic agent may be capable ofbinding to the GPC5 binding agent.

The skilled person will be aware of numerous possible mechanisms bywhich suitable therapeutic agents can be directed to a target cell via aGPC5 binding agent.

The therapeutic agent may comprise a cell or molecule of the immunesystem. For example, an anti-GPC5 antibody bound to a target cell may becapable of recruiting various effector mechanisms of the immune systemto attack that cell. These include cellular mechanisms, such asantibody-directed cell-mediated cytotoxicity, which is mediated bypolymorphonuclear cells, mononuclear cells and K cells, as well asmolecular mechanisms such as the complement cascade.

Alternatively, the therapeutic agent may comprise a molecule capable ofdirectly killing or inhibiting proliferation of the cell, such as atoxin or drug. This approach includes the use of precursor moleculescapable of being converted to toxin or drug molecules by action of anenzyme expressed by the cell or associated with the GPC5 binding agent.An example of such a method is often referred to as ADEPT therapy (seebelow).

In yet further alternative embodiments the therapeutic agent maycomprise a vector, such as a viral vector, comprising nucleic acidencoding a toxic or inhibitory agent to be synthesised within the cell.In such cases the agent encoded by the vector may itself be a GPC5antagonist as described elsewhere in this specification.

These approaches may be used individually or in combination. Othersuitable embodiments will be apparent to the skilled person.

A GPC5 binding agent may (but need not) have GPC5 antagonist activity inits own right.

A GPC5 antagonist is an agent which inhibits either the activity of GPC5or the expression of functional GPC5 on the cell surface. Thus a GPC5antagonist may inhibit cellular proliferation by directly blocking theproliferative effects of GPC5.

GPC5 antagonists which affect GPC5 activity (rather than expression) aretypically GPC5 binding agents which prevent or inhibit the GPC5 proteinfrom exerting its physiological activity, e.g. by blocking GPC5 frombinding to a ligand, receptor, and/or co-receptor. For example, anantibody, peptide, small molecule or the like which performs any one ofthese functions may be regarded as a GPC5 antagonist as well as a GPC5binding agent.

GPC5 antagonists which inhibit GPC5 expression may act at any one of anumber of points in the generation of mature GPC5 protein. For example,the antagonist may inhibit transcription of the GPC5 gene, processing ofGPC5 pre-mRNA, translation of GPC5 mRNA into protein, glycosylation ofGPC5 (i.e. addition of carbohydrate residues to the GPC5 core protein)or processing of the carbohydrate chains into mature heparan sulphate(HS) chains.

Preferred antagonists of GPC5 expression comprise nucleic acid sequencescomplementary to the sequence of GPC5 mRNA or pre-mRNA. These includeantisense RNA, dsRNA molecules (including RNAi and siRNA), andribozymes.

As set out above, GPC5 antagonists may sensitise target cells tocytotoxic agents. Thus the method may comprise the further step ofcontacting the cell with a cytotoxic agent, wherein the GPC5 antagonistincreases the sensitivity of the cell to the cytotoxic agent. This maybe particularly useful in treatment of cancer patients whose cells havebecome resistant to a chemotherapeutic agent; the GPC5 antagonist may beused in combination with the chemotherapy to increase the efficacy ofthe chemotherapeutic agent.

Methods of the invention have applications both in vitro and in vivo,but as will be clear from the above, preferred aspects involve theadministration of GPC5 antagonists or binding agents to subjectssuffering from cancer, in order to inhibit proliferation of cancercells.

Thus the invention further provides a method of treating cancer,comprising administering a GPC5 antagonist or a GPC5 binding agent to asubject suffering therefrom.

The invention further provides a GPC5 antagonist or a GPC5 binding agentas described herein for use in a method of medical treatment.

Also provided is use of a GPC5 antagonist or a GPC5 binding agent asdescribed herein in the preparation of a medicament for the treatment ofcancer.

In all of the therapeutic methods and compositions described herein, theGPC5 antagonists and binding agents may be used alone or in combinationwith other therapeutic agents, including cytotoxic agents (see above).

In a further aspect, the invention provides a method of determining thesusceptibility of a cancer to treatment with a GPC5 antagonist orbinding agent, comprising determining the presence, absence or level ofexpression of GPC5 in a cell from said cancer. Additionally oralternatively; the method may comprise the step of determining thepresence, absence or degree of chromosomal amplification at 13q31, e.g.determining the genomic copy number of the GPC5 gene. Additionally oralternatively the method may comprise the step of determining thepresence, absence or degree of WT1 expression in the cell. The cell maypreviously have been found to express WT1 inappropriately or tooverexpress WT1. Determinations may be qualitative, quantitative orsemi-quantitative.

The method will typically be performed in vitro using a sample isolatedfrom a subject suffering from the cancer in question. The sample maycomprise whole cells or cell extracts and may be derived from a biopsy,a body fluid such as blood, or any other suitable sample suspected orknown to contain one or more cancer cells. The method may comprise thestep of isolating the sample from the subject.

Typically the method will comprise the step of contacting the samplewith a GPC5 binding agent, which in this aspect of the invention may becapable of binding to GPC5 protein, DNA or RNA. The method may furthercomprise the step of determining the amount of binding agent bound toGPC5 and correlating the results obtained with a likelihood that thecancer will be susceptible to treatment with a GPC5 antagonist orbinding agent.

The presence or level of free circulating GPC5 (i.e. GPC5 protein notbound to a cell surface via a GPI anchor) may also serve as a marker fora cancer in which GPC5 is overexpressed. Thus in a further aspect theinvention provides a method of screening for the presence of a cancer ina patient, the method comprising contacting a sample derived from thepatient with a GPC5 binding agent. Preferably the sample is a sample ofa body fluid, such as whole blood, serum, plasma urine, etc.

The method may further comprise the step of determining the amount ofbinding agent bound to GPC5 and correlating the results obtained with alikelihood that the patient has cancer.

The inventors have also found that overexpression of GPC5 in breastcancer samples correlates well with the stage of that cancer. Inparticular, tumours which overexpress GPC5 are significantly more likelyto be stage 3 tumours than stage 1 or stage 2 tumours. This implies thatGPC5 can be used as a prognostic marker for breast cancer.

Therefore the invention provides a method for determining a prognosisfor a patient with breast cancer comprising assigning a prognosis to thepatient based on the expression levels of GPC5 in a breast tumour fromthat patient.

The method typically comprises determining the presence, absence ordegree of expression of GPC5 in a sample containing breast cancer cells.The method is typically performed in vitro using a sample isolated fromthe patient, although in vivo methods may also be envisaged. The samplemay be contacted with a GPC5 binding agent capable of binding to GPC5mRNA or protein.

“Prognosis” is intended in its most general sense, and may bequantitative or qualitative. It may be expressed in general terms, suchas a “good” or “bad” prognosis, and/or in terms of likely clinicaloutcomes, such as duration of disease free survival (DFS), likelihood ofsurvival for a defined period of time, and/or probability of distantmetastasis within a defined period of time. Quantitative measures ofprognosis will generally be probabilistic. Additionally oralternatively, and especially for communicating the prognosis to orbetween medical practitioners, the prognosis may be expressed in termsof another indicator of prognosis, such as the NPI scale.

In general, a patient with a ‘good prognosis’ tumour would probably betreated with a conventional treatment regimen. A patient with a ‘poorprognosis’ tumour might be treated with an alternative or moreaggressive regimen. The ‘poor prognosis’ patient would usually not haveto wait for the conventional treatment regimen to fail before movingonto the more aggressive one. Furthermore, having an understanding ofthe likely clinical course of the disease allows a patient to prepare arealistic plan for future, which is an important social aspect of cancertreatment.

For the avoidance of doubt, the term “determining” need not implyabsolute certainty in prognosis. Rather, the expression levels of GPC5in a tumour will generally be indicative of the likely prognosis of thepatient.

Those patients whose tumours are found to overexpress GPC5 are likely tohave stage 3 tumours, which may result in them being assigned a poorprognosis. It will be understood, though that GPC5 will not necessarilybe the sole marker used in determination of prognosis. Rather, it may beused in combination with other prognostic makers to assist in reaching adetailed prognosis.

The inventors envisage that GPC5 expression may also be used to monitorthe progress (e.g. success or failure) of a treatment for a cancerpreviously found to express GPC5. Such methods typically involvemonitoring GPC5 expression in cells of the cancer. This may involvedetermining the level of expression of GPC5 within cells of the cancer.A reduction in the level of GPC5 expression over time may be taken as anindication that the treatment is effective. Additionally oralternatively, the method may involve determining the number or densityof cells in a given sample expressing or overexpressing GPC5. This mayassist in determining whether the size of the tumour is being reduced,so giving an indication of whether the treatment is having the desiredeffect of reducing tumour size.

The method will typically involve comparing the results obtained withresults of an equivalent assay performed for the same patient beforetreatment, and/or at an earlier stage of treatment.

The method typically comprises determining the presence, absence ordegree of expression of GPC5, or the number or density of cellsexpressing or overexpressing GPC5, in a sample containing breast cancercells from the patient. As with other methods described herein, it istypically performed in vitro using a sample isolated from the patient,although in vivo methods may also be envisaged. The sample is typicallycontacted with a GPC5 binding agent capable of binding to GPC5 mRNA orprotein.

In a further aspect the invention further provides methods of screeningwhich may be used to identify therapeutic agents. In particular theinvention provides a method of screening for an agent capable of killingor inhibiting proliferation of a target cell, comprising the steps of:

(i) contacting GPC5 protein with one or more candidate substances;(ii) selecting one or more candidate substances based on their abilityto bind GPC5 protein;(iii) contacting said one or more selected substances with a targetcell; and(iv) determining the effect of said selected substance(s) onproliferation of said cell.

The cell typically expresses GPC5, and preferably inappropriatelyexpresses or overexpresses GPC5. The cell may naturally express GPC5(e.g. it may be derived from a cancer in which GPC5 is expressed oroverexpressed) or it may have been engineered to express GPC5, e.g. bytransformation with a vector comprising nucleic acid encoding GPC5. Thecell may previously have been found to inappropriately express oroverexpress WT1.

The method may comprise the step of further selecting one or moresubstances found to inhibit proliferation of the target cell. Theselected substances may be subjected to one or more rounds ofmodification, to increase activity and/or suitability for in vivoadministration and re-tested for the ability to inhibit cellularproliferation. Suitable substances may be formulated for therapeuticadministration, e.g. as a pharmaceutical composition.

In the various methods described above, the target cell or cancer celltypically overexpresses GPC5 mRNA and/or GPC5 protein. The cell may alsocarry a chromosomal amplicon comprising part or all of the 13q31 region;i.e. the cell may carry more than the normal two genomic copies of theGPC5 gene. In preferred embodiments, though, the cell overexpresses GPC5but does not carry any amplification of that chromosomal region. Thetarget cell is typically a cancer cell, and preferably one whichoverexpresses GPC5.

Cancers previously found to carry 13q31-32 amplicons include examples ofrhabdomyosarcomas, including both embryonal and alveolar RMS, lymphomasincluding follicular lymphoma, mantle cell lymphoma and primarycutaneous B-cell lymphoma, non-small cell lung cancer, bladder cancer, asmall proportion of breast cancers, neuroglial tumours includingmalignant peripheral nerve sheath tumours, squamous cell carcinoma ofthe head and neck, chronic myeloid leukemia, leiomyosarcoma,liposarcoma, malignant fibrous histocytoma of bone and soft tissues,(See Gordon et al., 2000, Yu et al., 2003, and references citedtherein).

In view of the finding that GPC5 overexpression occurs in the absence ofchromosomal amplification in RMS, prostate cancer and breast cancer, itis likely that examples of the above described cancer types will alsooverexpress GPC5 without 13q31 amplification and so any of the above areenvisaged as suitable for treatment by the methods and compositionsdescribed in this specification.

GPC5 expression appears to be regulated (at least in part) by WT1.Accordingly, cancers which show inappropriate expression (e.g.overexpression) of WT1 may also be suitable for treatment by the methodsdescribed. These include leukaemias and a wide range of solid tumoursincluding prostate, breast and lung, as well as thyroid, testicular andovarian carcinomas, melanoma and mesothelioma (Scharnhorst, V., van derEb, A. J., and Jochemsen, A. G. (2001) Gene 273, 141-161).

WT1 expression has also been implicated in the resistance of cancercells to chemotherapeutic agents. Thus, cancers which are resistant totreatment with one or more cytotoxic agents may also be suitable fortreatment according to the present invention. The cells may have beenresistant ab initio or may have developed a resistance over the courseof treatment. Typically such cells will be characterised byoverexpression of WT1 and GPC5.

In addition, improved efficacy of GPC5 antagonists in increasingchemosensitivity may be achieved by concomitant down regulation of WT1using WT1 antagonists. Preferred WT1 antagonists comprise nucleic acidsequences complementary to the sequence of WT1 mRNA or pre-mRNA. Theseinclude antisense RNA, dsRNA molecules (including RNAi and siRNA), andribozymes.

DESCRIPTION OF THE DRAWINGS

FIG. 1: FISH results using BACs spanning the amplified 13q31q32 regionin two primary RMS samples and the cell line K562. The minimal region ofamplification is defined by RP11-51a2 at the centromeric end and by GPC6Taqman at the telomeric end which shows no amplification. Therefore, theregion of minimal amplification flanks the GPC5 gene but does not extendinto the next telomeric gene GPC6. The gene C13ORF25A, locatedproximally centromeric to GPC5, has been suggested to be implicated inlymphoma (33).

FIG. 2A: Log distribution of GPC5 expression relative to normal muscle;left (Embryonal), right (Alveolar).

FIG. 2B: Expression of C13ORF25A in rhabdomyosarcoma relative to normalmuscle. Bars marked “A” indicate samples which show genomicamplification of C13ORF25A.

FIG. 3 shows the effect of GPC5 antisense oligonucleotides on GPC5 mRNAlevels and cell survival in K562 cells. Panel A shows data for 13representative GPC5-targeted oligonucleotides relative to the controloligonucleotide 15770. Panels B and C show dose-response curves for oneactive oligonucleotide (276107) relative to a different controloligonucleotide, designated 276124.

FIG. 4A shows the level of GPC5 mRNA expression in 18 prostate cancersamples and 6 benign prostate hyperplasia samples, as compared to normalprostate. FIG. 4B shows the overall difference in GPC5 expressionbetween prostate cancer and benign prostatic hyperplasia.

FIG. 5A shows GPC5 mRNA expression in breast cancer samples relative tonormal breast biopsy tissue. FIG. 5B shows the number of tumours ofstage 1, 2 or 3 which overexpress GPC5 relative to normal breast tissuein a sample of 44 breast tumours.

FIG. 6 shows the effect of WT1 antisense oligonucleotides on WT1 andGPC5 mRNA levels in K562 cells, both 24 and 48 hours after treatment,suggesting that WT1 may regulate GPC5 expression.

FIG. 7 shows upregulation of both WT1 and GPC5 mRNA in PC3M tumorstreated with docetaxol.

FIG. 8 shows the effect of GPC5 overexpression in the RMS cell lineT91-95. A: Difference in proliferation between 5 GPC5 overexpressingcolonies and 5 mock-transfected control colonies. B: Difference inexpression of GPC5 as measured by TaqMan assay between GPC5overexpressing colonies and control colonies. C: Western Blot of an InVitro Translation; Lane 1 water (negative control), Lane 2 pCMV-TNT-GPC5(63 Kda), Lane 3 Luciferase (61 Kda positive control). Protein wastranslated so as to incorporate biotin labelled lysine residues. Colourwas developed using streptavidin-alkaline phosphatase and appropriatecolorimetric reagents.

DETAILED DESCRIPTION OF THE INVENTION Glypicans

The glypicans are cell surface heparan sulphate proteoglycan (HSPG)molecules which consist of a protein core carrying heparan sulphate (HS)glycosaminoglycan chains and linked to the plasma membrane of the cellthrough a GPI anchor. For general reviews see Perrimon and Bernfield(2000) Nature 404: 725-728 and Selleck (1999) Am J Hum Genet 64:372-377.

The HS chains themselves are attached to the serine residues ofconsensus SGXG “glycanation” sites. They each consist of atetrasaccharide linker (-GlcA-Gal-Gal-Xyl) to which is added a linearpolymer of a repeating disaccharide unit made up of GlcNac and GlcA.After the chains have been synthesised, they are subjected to processingin the Golgi by the enzymes N-deacetylase/N-sulphotransferase (NDST),uronosyl C5-epimerase, 2-O-sulphotransferase (2-OST),6-O-sulphotransferase (6-OST) and 3-O-sulphotransferase (3-OST).

The HSPGs are increasingly thought to play specific roles in cellsignalling. Some HSPGs may act as co-receptors for growth factorsignalling (see the reviews by Perrimon and Bernfield, and Selleck,above). Glypican-3 has been shown to co-immunoprecipitate with FGF2 andBMP-7 (Int J Cancer 2003 103:455-65), while glypican-1 has been shown toco-immunoprecipitate with FGF2 and HB-EGF and to increase the growthstimulatory action of these growth factors (J Clin Invest 1998 102(9)1662-73). Glypican-4 modulates the function of FGF2 by suppressing itsgrowth stimulatory properties (Dev Dyn 219(3):353-67).

The mRNA and protein sequence of human GPC5 is found in Veugelers et al“Characterization of Glypican-5 and chromosomal localization of humanGlypican-5; new member of glypican gene family” Genomics 199740(1):24-30. The genomic structure of the gene is described in Veugelerset al “A 4MB BAC/PAC contig+complete genomic structure of the GPC5/GPC6gene cluster on chromosome 13q32” Matrix Biol 2001 20(5-6):375-85. TheGenBank reference sequence accession number is AF001462 (gi:3015541) formRNA and NP004457 for protein. Homologous proteins have also beenidentified in mouse (NP 780709) and rat (XP 224489) having proteinsequence identity of 82% and 87% respectively to the human sequence.This is greater than the next most homologous gene within the humangenome GPC3 (NP 004475) which has a protein sequence identity of 46%with human GPC5.

“GPC5 protein” is used herein as a general term to include both the coreprotein and/or its associated heparan sulphate chains. References to theprotein should be construed accordingly.

Cells

The term “cancer cell” is used throughout this specification to refer toany transformed cell including cells from cancers and tumours occurringin vivo, as well as laboratory cell lines adapted to continuous culture.Such cell lines typically display characteristics such as unlimitedcapacity for in vitro replication, loss of contact inhibition, abilityto form tumours in animals, etc. They may historically be derived from acancer, or may have been transformed in the laboratory.

The cell may be of any suitable species, although mammalian cells arepreferred. Particularly preferred are human and rodent (e.g. mouse orrat) cells.

Target cells of the various methods described typically overexpressGPC5. The cells may have been engineered to overexpress GPC5, e.g. bytransformation with a vector comprising nucleic acid encoding GPC5, ormay naturally overexpress GPC5, in that they overexpresses GPC5 withouthaving been deliberately manipulated to do so.

A cell is considered to overexpress GPC5 if it shows a higher level ofRNA or protein than normally found in that cell type. A tumour cell maybe considered to overexpress GPC5 if it shows a level of expressiongreater than that found in a corresponding cell type from which thetumour is thought to be derived. For example, RMS cells can be comparedto normal muscle cells or muscle precursor cells, breast cancer cells tohealthy breast tissue or normal breast epithelial cells and prostatecancer cells to healthy prostate tissue or normal prostate epithelium.

Thus a cell type which does not normally express GPC5 may be consideredto overexpress, or inappropriately express, GPC5 if it shows adetectable level of GPC5 expression. A cell which would normally showdetectable GPC5 expression may be considered to overexpress GPC5 if itshows double the normal level of RNA or protein for that cell type, morepreferably 5 times, 10 times, 50 times or 100 times the normal level forthat cell type. The brain is the only normal human adult tissue whichhas been reported to display detectable expression of GPC5.

The same considerations, mutatis mutandis, apply to determining whetherwhole tissues, body fluids etc. display elevated levels of GPC5.

WT1 is normally only expressed in specific cell types in kidney, gonads,haematopoietic and nervous system, and mesothelium (Reddy, J. C., andLicht, J. D. (1996) Biochim Biophys Acta 1287, 1-28). Overexpression ofWT1 in these tissues or cancers derived therefrom may be determined asdescribed above for GPC5. Expression in other cell types may beconsidered inappropriate.

Antagonists

The term “GPC5 antagonist” encompasses two different classes of agent.

An “antagonist of GPC5 activity” is an agent which prevents the matureGPC5 protein from exerting its normal function when expressed at thecell surface. Typically these will be binding agents for GPC5 proteinwhich are capable of binding to GPC5 protein (core protein and/or HSPGchains) and inhibiting its pro-proliferative function.

An “antagonist of GPC5 expression” is an agent capable of inhibiting orblocking expression of the mature protein at the cell surface, althoughit will be appreciated that the ultimate effect of such agents is alsoto inhibit GPC5 function or activity.

GPC5 antagonists may therefore be identified by screening candidatecompounds or substances for the ability to bind GPC5. Suitable assaymethods are described below.

Those candidates which show suitable binding may be screened(subsequently or in parallel) for their ability to inhibit proliferationof a cell overexpressing GPC5. Suitable cells include those whichnaturally overexpress GPC5 (such as cell lines derived fromGPC5-overexpressing cancers), as well as those engineered to overexpressGPC5 (e.g. by transformation with a vector encoding GPC5).

By contrast, antagonists of GPC5 expression typically comprise nucleicacid molecules capable of hybridising to GPC5 genomic DNA, precursormRNA, mRNA, or cDNA, which may be single stranded or double stranded.Such modulators include antisense RNA or DNA, triple helix-formingmolecules, RNAi, siRNA and ribozymes. (Such antagonists may also beconsidered to be GPC5 binding agents as described below.) Nucleic acidscomprising chemically modified nucleotides (such as locked nucleicacids, or propynyl, methyl or G-clamped pyrimidine nucleotides) as wellas nucleic acid analogues having modified sugar residues (e.g.2′-O-methyl and 2′-methoxyethyl modifications) or backbone structure(e.g. by incorporation of phosphoramidite or morpholino linkages, orpeptide nucleic acids (PNAs)) are also included within this definition.For more details of these modifications and antisense techniques ingeneral, see Dean and Bennett (2003) Oncogene 22: 9087-9096 andreferences cited therein.

Antisense oligonucleotides hybridise with complementary sequences of RNAgenerally by Watson-Crick base pairing. The resultant double strandedcomplex prevents translation of the message into protein product eitherby steric blocking at the ribosome or activation of RNase H that cleavesthe RNA strand of the duplex. With respect to antisense DNA,oligodeoxy-ribonucleotides derived from the translation initiation site,e.g. between the −10 and +10 regions of the target gene nucleotidesequence of interest, are preferred.

In using antisense genes or partial gene sequences to down-regulate geneexpression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene.

The complete sequence corresponding to the coding sequence need not beused. For example fragments of sufficient length may be used. It is aroutine matter for the person skilled in the art to screen fragments ofvarious sizes and from various parts of the coding sequence to optimisethe level of antisense inhibition. It may be advantageous to include theinitiating methionine ATG codon, and perhaps one or more nucleotidesupstream of the initiating codon. A further possibility is to target aconserved sequence of a gene, e.g. a sequence that is characteristic ofone or more genes, such as a regulatory sequence.

The sequence employed may be 500 nucleotides or less, possibly about 400nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100nucleotides. It may be possible to use oligonucleotides of much shorterlengths, 14-23 nucleotides, although longer fragments, and generallyeven longer than 500 nucleotides are preferable where possible.

It may be preferable that there is complete sequence identity in thesequence used for down-regulation of expression of a target sequence,and the target sequence, though total complementarity or similarity ofsequence is not essential. One or more nucleotides may differ in thesequence used from the target gene. Thus, a sequence employed in adown-regulation of gene expression in accordance with the presentinvention may be a wild-type sequence (e.g. gene) selected from thoseavailable, or a mutant, derivative, variant or allele, by way ofinsertion, addition, deletion or substitution of one or morenucleotides, of such a sequence. The sequence need not include an openreading frame or specify an RNA that would be translatable. It may bepreferred for there to be sufficient homology for the respectiveanti-sense and sense RNA molecules to hybridise. There may be downregulation of gene expression even where there is about 5%, 10%, 15% or20% or more mismatch between the sequence used and the target gene.

Double stranded RNA (dsRNA) has been found to be even more effective ingene silencing than antisense strands alone (Fire A. et al Nature, Vol391, (1998)). dsRNA mediated silencing is gene specific and is oftentermed RNA interference (RNAi). RNA interference is a two step process.First, dsRNA is cleaved within the cell to yield short interfering RNAs(siRNAs) of about 21-23 nt length with 5′ terminal phosphate and 3′short overhangs (˜2 nt) The siRNAs target the corresponding mRNAsequence specifically for destruction (Zamore P. D. Nature StructuralBiology, 8, 9, 746-750, (2001) RNAi may be also be efficiently inducedusing chemically synthesized siRNA duplexes of the same structure with3′-overhang ends (Zamore P D et al Cell, 101, 25-33, (2000)). SyntheticsiRNA duplexes have been shown to specifically suppress expression ofendogenous and heterologeous genes in a wide range of mammalian celllines (Elbashir S M. et al. Nature, 411, 494-498, (2001)). See also Fire(1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490,Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001)Chem. Biochem. 2: 239-245.

Ribozymes are enzymatic RNA molecules capable of catalysing the specificcleavage of RNA. (For a review, see Rossi, J., 1994, Current Biology 4:469-471). The mechanism of ribozyme action involves sequence specifichybridisation of the ribozyme molecule to complementary target RNA,followed by an endonucleolytic cleavage. The composition of ribozymemolecules must include one or more sequences complementary to the targetprotein mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see U.S. Pat. No.5,093,246, which is incorporated by reference herein in its entirety. Assuch, within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyseendonucleolytic cleavage of RNA sequences encoding target proteins.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the molecule of interest for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short sequences of between 15 and 20 ribonucleotidescorresponding to the region of the target protein gene containing thecleavage site may be evaluated for predicted structural features, suchas secondary structure, that may render the oligonucleotide sequenceunsuitable. The suitability of candidate sequences may also be evaluatedby testing their accessibility to hybridise with complementaryoligonucleotides, using ribonuclease protection assays.

Nucleic acid molecules to be used in triplex helix formation for theinhibition of transcription should be single stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides must bedesigned to promote triple helix formation via Hoogsteen base pairingrules, which generally require sizeable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGC⁺triplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementary to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesised in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

WT1 antagonists are agents which prevent WT1 exerting its function as atranscription factor. As it is an intracellular protein, preferred WT1antagonists are those which interfere with WT1 expression. Particularlypreferred WT1 antagonists comprise nucleic acid sequences complementaryto the sequence of WT1 mRNA or pre-mRNA. These include antisense RNA,dsRNA molecules (including RNAi and siRNA), and ribozymes. For exemplaryWT1 sequences, see GenBank accession numbers NM_(—)000378.2,NM_(—)024426.2, NM_(—)024425.1, NM_(—)024424.1. All accession numberscited in this application are taken from GenBank release 140.0, updated20 Feb. 2004.

Binding Agents

In most aspects of the invention, GPC5 binding agents are referred to inthe context of agents capable of binding to GPC5 core protein and/or itsassociated heparan sulphate chains. However in some aspects of theinvention, such as the diagnostic methods described below, agentscapable of binding to nucleic acid encoding GPC5, and in particular toGPC5 pre-mRNA, mRNA, or cDNA derived therefrom, may also be consideredto be GPC5 binding agents.

In preferred embodiments the binding agents which are used may beregarded to constitute a specific binding pair with GPC5. The term“specific binding pair” may be used to describe a pair of moleculescomprising a specific binding member (sbm) and a binding partner (bp)therefor which have particular specificity for each other and which innormal conditions bind to each other in preference to binding to othermolecules. Examples of specific binding pairs are antigens andantibodies, ligands (such as hormones, etc.) and receptors,avidin/streptavidin and biotin, lectins and carbohydrates, andcomplementary nucleotide sequences.

By “specific” is meant that the particular binding sites of the agentwhich interact with GPC5 will not show any significant binding tomolecules other than GPC5 which are likely to be encountered by thebinding agent (e.g. other molecules in an assay or on a given cellsurface). For example the interaction between the binding agent and GPC5may have a K_(D) of the order of 10⁻⁶ to 10⁻⁹M⁻¹ or smaller.

The binding agent may bind to the protein core or the HS chains of theGPC5 molecule, but in preferred embodiments bind to the protein core,preferably to hydrophilic regions of the core, e.g. to part or all ofthe sequence CKSYTQRVVGNGIKAQ.

The binding agent may be a protein or polypeptide of 50 amino acids insize or greater, or a peptide of up to 50 amino acids in length.Typically a peptide will be from 5 to 50 amino acids in length, moretypically 10 to 20 amino acids in length. Alternatively the bindingagent may be a small molecule e.g. of 1000 Da or less, preferably 750 Daor less, preferably 500 Da or less.

Antibodies are preferred examples of binding agents. Thus preferredassay formats for diagnosis are immunological assays including ELISAassays, and immunohistochemistry, which may be carried out on wholecells or tissue sections, other forms of immunostaining for FACSanalysis, confocal microscopy or the like, which may be carried out onsingle cells or populations of dispersed cells, and immunoblotting,which is suitable for analysis of cell extracts.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. The term “antibody” is therefore usedherein to encompass any molecule comprising the binding fragment of anantibody. Examples of binding fragments are (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains; (ii) the Fd fragmentconsisting of the VH and CH1 domains; (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VHdomain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding member (Bird et al, Science, 242, 423-426, 1988; Hustonet al, PNAS USA, 85, 5879-5883, 1988).

Diagnostic Methods and Other Assays

As well as being useful as GPC5 antagonists and targeting agents,binding agents for GPC5 may also be used to detect the presence of GPC5in biological samples. This has a number of applications within thescope of the invention.

The binding agents described herein may be used to assess thesusceptibility of a particular cancer to treatment with GPC5 antagonistsor binding agents by assessing the level of expression of GPC5 in thatcancer. A cancer found to overexpress or have an elevated level of GPC5may be treatable by means of the methods and compositions describedherein.

The method typically comprises contacting a sample with a GPC5 bindingagent, the sample having been isolated from a subject suffering from thecancer in question. The sample may comprise one or more whole cells orextracts of cells, and may be derived from any suitable biologicalsample suspected or known to contain one or more cancer cells. Examplesinclude tissue samples (e.g. biopsies) and samples of body fluid, e.g.blood, serum or plasma.

The binding agent may detect expression of either GPC5 protein or mRNA.

In preferred embodiments the method further comprises comparing thelevel of GPC5 expression with that found in one or more referencesamples, which may be pre-determined. Suitable reference samples includesamples of the same tissue type, or a comparable tissue type, as thatfrom which the cancer or suspected cancer is derived. The referencesamples may be obtained from the same individual as the test sample, orfrom different individuals. The reference samples may also includesamples of cancer cells of known type, optionally of known GPC5expression level, which may serve as positive controls.

The sample may be derived from a cancer previously identified toinappropriately express or overexpress WT1. Alternatively the method mayfurther comprise the step of determining the level of expression of WT1to see whether combined therapy with one or more WT1 antagonists wouldbe beneficial.

The invention also provides methods of determining whether a subject issuffering from a cancer characterised by overexpression of GPC5, themethod comprising contacting a sample from the subject with a GPC5binding agent. Preferably the method comprises determining the level ofcirculating free (i.e. not cell-associated) GPC5. In such cases, thesample is preferably blood, serum or plasma, and the binding agentdetects GPC5 protein.

Again, the results obtained may be compared with suitable positiveand/or negative control samples to arrive at an indication of thesubject's clinical status.

Methods for determining the concentration of analytes in samples fromindividuals are well known in the art and readily adapted by the skilledperson in the context of the present invention to determine expressionof GPC5 protein or mRNA as appropriate. Such assays may allow aphysician to optimise the treatment of a disorder and, thus, the methodsdescribed allow for planning of appropriate therapy, permittingstream-lining of treatment by targeting those most likely to benefit.

Assay methods for determining, the concentration of protein markerstypically employ binding agents having binding sites capable ofspecifically binding to protein markers, or fragments thereof, orantibodies in preference to other molecules. Examples of binding agentsinclude antibodies, receptors and other molecules capable ofspecifically binding the analyte of interest. Conveniently, the bindingagents are immobilised on a solid support, e.g. at defined, spatiallyseparated locations, to make them easy to manipulate during the assay.

The sample is generally contacted with the binding agent(s) underappropriate conditions which allow the analyte in the sample to bind tothe binding agent(s). The fractional occupancy of the binding sites ofthe binding agent(s) can then be determined either by directly orindirectly labelling the analyte or by using a developing agent oragents to arrive at an indication of the presence or amount of theanalyte in the sample. Typically, the developing agents are directly orindirectly labelled (e.g. with radioactive, fluorescent or enzymelabels, such as horseradish peroxidase) so that they can be detectedusing techniques well known in the art. Directly labelled developingagents have a label associated with or coupled to the agent. Indirectlylabelled developing agents may be capable of binding to a labelledspecies (e.g. a labelled antibody capable of binding to the developingagent) or may act on a further species to produce a detectable result.Thus, radioactive labels can be detected using a scintillation counteror other radiation counting device, fluorescent labels using a laser andconfocal microscope, and enzyme labels by the action of an enzyme labelon a substrate, typically to produce a colour change. In furtherembodiments, the developing agent or analyte is tagged to allow itsdetection, e.g. linked to a nucleotide sequence which can be amplifiedin a PCR reaction to detect the analyte. Other labels are known to thoseskilled in the art are discussed below. The developing agent(s) can beused in a competitive method in which the developing agent competes withthe analyte for occupied binding sites of the binding agent, ornon-competitive method, in which the labelled developing agent bindsanalyte bound by the binding agent or to occupied binding sites. Bothmethods provide an indication of the number of the binding sitesoccupied by the analyte, and hence the concentration of the analyte inthe sample, e.g. by comparison with standards obtained using samplescontaining known concentrations of the analyte.

In alternative embodiments, the analyte can be tagged before applying itto the support comprising the binding agent.

Preferred formats are ELISA assays and immunostaining (e.g.immunohistochemistry).

There is also an increasing tendency in the diagnostic field towardsminiaturisation of such assays, e.g. making use of binding agents (suchas antibodies or nucleic acid sequences) immobilised in small, discretelocations (microspots), and/or as arrays on solid supports or ondiagnostic chips. These approaches can be particularly valuable as theycan provide great sensitivity (particularly through the use offluorescent labelled reagents), require only very small amounts ofbiological sample from individuals being tested and allow a variety ofseparate assays can be carried out simultaneously. This latter advantagecan be useful as it provides an assay employing a plurality of analytesto be carried out using a single sample. Examples of techniques enablingthis miniaturised technology are provided in WO84/01031, WO88/1058,WO89/01157, WO93/8472, WO95/18376/WO95/18377, WO95/24649 and EP 0 373203 A. Thus, in a further aspect, the present invention provides a kitcomprising a support or diagnostic chip having immobilised thereon aplurality of binding agents capable of specifically binding differentprotein markers or antibodies, optionally in combination with otherreagents (such as labelled developing reagents) needed to carrying outan assay. In this connection, the support may include binding agentsspecific for analytes such as vimentin, e.g. as disclosed in U.S. Pat.No. 5,716,787.

Such assay methods may also be used to screen for binding agents capableof binding to GPC5 protein. Candidate agents identified by such screensmay be subjected to one or more rounds of modification and re-testing inorder to identify further agents having improved properties. The skilledperson will be aware of numerous suitable screening methods and will beable to design appropriate protocols for identification of candidatebinding agents.

Alternatively the binding agent may be a nucleic acid molecule capableof binding to mRNA or precursor mRNA. Thus mRNA or precursor mRNAencoding GPC5 may be detected by hybridisation with a probe having asuitable complementary sequence, e.g. by Northern blotting or in situhybridisation. Such protocols may use probes of at least about 20-80bases in length. The probes may be of 100, 200, 300, 400 or 500 bases inlength or more. Binding assays may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989or later editions).

Alternatively, conventional RT PCR procedures (including quantitativePCR procedures) may be used to analyse the presence or amount of mRNA orprecursor mRNA in a given sample. A suitable primer having at least 15to 20 bases complementary to the GPC5 mRNA or precursor mRNA sequencewill typically be used to prime cDNA synthesis. Subsequently, a segmentof the cDNA is amplified in a PCR reaction using a pair of nucleic acidprimers. The skilled person will be able to design suitable probes orprimers based on the publicly available sequence data for GPC5 (seeabove).

Whether it is a protein, peptide, small molecule or nucleic acid, thebinding agent may also act as an antagonist of GPC5.

Pharmaceutical Compositions

GPC5 antagonists and binding agents can be formulated in pharmaceuticalcompositions. These compositions may comprise, in addition to one of theabove substances, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes or topical application.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,small molecule or other pharmaceutically useful compound according tothe present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Suitable carriers, adjuvants, excipients, etc. can be found in standardpharmaceutical texts, for example Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook ofPharmaceutical Excipients, 2nd edition, 1994.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be producedin the target cells by expression from an encoding gene introduced intothe cells, eg in a viral vector (a variant of the VDEPT technique—seebelow). The vector could be targeted to the specific cells to betreated, or it could contain regulatory elements which are switched onmore or less selectively by the target cells. The targeting method mayitself make use of the expression of GPC5 on the surface of the targetcells.

Alternatively, the agent could be administered in a precursor form, forconversion to the active form by an activating agent produced in, ortargeted to, the cells to be treated. This type of approach is sometimesknown as ADEPT or VDEPT; the former involving targeting the activatingagent to the cells by conjugation to a cell-specific antibody, while thelatter involves producing the activating agent, e.g. an enzyme, in avector by expression from encoding DNA in a viral vector (see forexample, EP-A-415731 and WO 90/07936).

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Expression of GPC5 in healthy adult human tissue is largely restrictedto the brain. Therefore when designing pharmaceutical and othercompositions for in vivo administration according to the invention, itmay be desirable to make use of components (and particularly activeingredients) which tend not to cross the blood-brain barrier.

The blood-brain barrier is most permeable to small (up to approx. 700Da) and/or lipophilic molecules such as water, carbon dioxide, oxygenand anaesthetic molecules, while being almost impermeable to plasmaproteins and non-lipid-soluble large organic molecules. It may thereforebe preferable that the compositions of the invention comprise GPC5antagonists and/or binding agents which are relatively hydrophilic,and/or above at least 1 kDa. Peptides and proteins may be preferred.Strategies for screening and design of GPC5 binding agents andantagonists may be designed accordingly.

Gene Therapy

Nucleic acids encoding modulators (antagonists) of GPC5 expression (e.g.antisense, RNAi, siRNA or ribozyme molecules) may be used in methods ofgene therapy. A construct capable of expressing such nucleic acid may beintroduced into cells of a recipient by any suitable means, such thatthe relevant sequence is expressed in the cells.

The construct may be introduced in the form of naked DNA, which is takenup by some cells of animal subjects, including muscle cells ofmammalians. In this aspect of the invention the construct will generallybe carried by a pharmaceutically acceptable carrier alone. The constructmay also formulated in a liposome particle, as described above.

Such methods of gene therapy further include the use of recombinantviral vectors such as adenoviral or retroviral vectors which comprise aconstruct capable of expressing a polypeptide of the invention. Suchviral vectors may be delivered to the body in the form of packaged viralparticles. The viral vectors may themselves be targeted to theappropriate cells via GPC5 binding agents.

Constructs of the invention, however formulated and delivered, will befor use in treating tumours in conjunction with therapy. The constructwill comprise the relevant nucleic acid linked to a promoter capable ofexpressing it in the target cells. The constructs may be introduced intocells of a human or non-human mammalian recipient either in situ orex-vivo and reimplanted into the body. Where delivered in situ, this maybe by for example injection into target tissue(s) or in the case ofliposomes, inhalation.

Gene therapy methods are widely documented in the art and may be adaptedfor use in the expression of the required sequence.

Materials and Methods Patient Samples and Cell Lines

Samples were collected from patients with a diagnosis of RMS from theRoyal Marsden NHS Trust or participating UKCCSG (United KingdomChildren's Cancer Study Group) centres around the time of firstdiagnosis. In addition, 22 samples were collected at the Universityhospital in Leuven, Belgium and two samples were collected fromUniversity hospital Dusseldorf. Samples were snap frozen and materialtaken adjacent to samples were taken to confirm high tumour content. Apathological diagnosis of RMS was made in the majority of cases by thepathological review committee of the MMT studies. In cases where therewas no central review of pathology, morphology and immunohistochemistryreports were examined to ensure RMS pathology. The diagnosis of ARMS wasconsistent with the current histopathological criteria whereby anyalveolar foci are sufficient to result in ARMS classification. Clinicaldata for the majority of tumours were obtained from the UKCCSG datacentre (Leicester, UK) otherwise data was collected directly fromparticipating hospitals. Only patients under the age of 21 were used insurvival analysis. The majority of patients were treated using the STOP(Societe Internationale de Oncologie Paediatrique) MMT89 (MalignantMesenchymal Tumour) protocol or the closely related MMT95 and MMT98protocols. Some patients were treated using local treatment protocolsthat were comparable to the MMT protocols. Tumour samples from breastand prostate cancer patients were snap frozen after removal. DNA and RNAwas extracted from samples as previously described (20). K562 cl.6cells, a subclone of the parent erythroleukaemia were kindly provided byProfessor Adrian Newland and Dr Xu-Rong Jiang, (The London HospitalMedical College, UK) and the RMS cell line T91-95 used in thetransfection studies was a kind gift from Jaclyn Biegel (Children'sHospital of Philadelphia).

Chromosomal Level Data on Genetic Imbalances and Differential Expression

Data from previous studies on RMS included comparative genomichybridization (CGH) analysis for genomic imbalances (n=127)(4-10)(unpublished data) and comparative expressed sequencehybridization (CESH) data for chromosomal level differential expression(n=45)(19, 20). These data were used here to compare the genomic changeswith differential expression relative to muscle at 13q31-q32.

Fluorescence In Situ Hybridization—FISH

Interphase fluorescence in situ hybridization (FISH) was performed asdescribed previously (21). We used touch preps from two ARMS primarysamples and the K562 cell line which possess a 13q31-32 amplicon. BAC(Bacterial Artificial Chromosome) clones spanning the 13q31-32 regionwere obtained from the Sanger Centre and included RP11-51a2, RP11-16n13,RP11-121J7, RP11-215 m7, RP11-57f10, RP11-169i15, RP11-210-3.

Real Time Quantification of GPC5/6 DNA and RNA Levels

Five sets of primers and probes were designed in order to measure theamount of genomic and mRNA copies of GPC5 and GPC6 (see Table 1). Allprimers and probes were designed in accordance with Applied Biosystems'TaqMan® standard requirements. Primers and a probe were designed withinintron 2 of the GPC5 gene to detect genomic copies of GPC5. To detectcopies of GPC6 primers and a probe were designed within exon 3. So as tocorrect for aneuploidy the gene GJB2 was chosen as an endogenouscontrol. GJB2 is a gene that is not believed to be involved intumorigenesis and located in a region of chromosome 13 (13q11) notfrequently altered in RMS. Primers and a probe were designed within exon1.

To measure the amount of mature mRNA copies of GPC5 and GPC6 the probewas designed across the exon 1-exon 2 boundary and the exon 7-exon 8boundary respectively. Applied Biosystems' Predeveloped GAPDH(Glyceraldehyde 3-phosphate dehydrogenase) was used as an endogenouscontrol. 25 μL multiplex PCR reactions were run using 2× UniversalTaqMan® Master Mix (Applied Biosystems Pt No. 4304437), theconcentration of primers and probes shown in Table 1 and 10 ng of DNA orcDNA. These samples were run in triplicate under standard operatingconditions on an ABI7700 SDS TaqMan® Machine (Applied Biosystems, CA).Limiting primer conditions were determined and template titrationsshowed that the comparative method was appropriate for both genomic andexpression reactions (data not shown). Thus, the amount of GPC5 and GPC6was measured relative to either normal genomic DNA in the case ofgenomic measurements and to normal muscle in the case of expressionmeasurements. Normal genomic DNA was extracted from the blood of ahealthy donor, normal muscle cDNA was produced from RNA extracted from apool of 11 normal muscle biopsies.

Statistics

All statistics tests were performed using the SPSS 10.0 package andtested to the 5% significance level. Failure free survival was definedas the time from diagnosis to relapse, progression, death or if eventfree to the date of last contact. Time to death was defined as the timefrom diagnosis to death or if event free to the date of last contact.

Real Time Quantification of WT1 mRNA Levels.

The primer pair and probe for the quantification of WT1 were designedusing the Primer Express program (Applied Biosystems) according to therecommended guidelines and as described above: WT1-Forward primer,5′-TACCCAGGCTGCAATAAGAGATATTTTAAG-3′, reverse primer,5′-CCTTTGGTGTCTTTTGAGCTGGTC-3′, and probe,5′-CACTGGTGAGAAACCATACCAGTGTGACTTCAAGGACT-3′. Each assay sample wasanalysed in triplicate as above, and multiplexed to facilitate themeasurement of gene expression levels relative to GAPDH (in vivo tumoursamples) or 18s ribosomal RNA expression (ribosomal RNA controlreagents, Applied Biosystems)(in vitro cell line samples) using thestandard curve method.

Polyclonal Antibody Production, Purification and Western Blotting

As no commercial antibody existed for GPC5 a custom polyclonal antibodywas raised to the epitope peptide H2N-CKSYTQRVVGNGIKAQ-COOH (16aa) byimmunising two rabbits (performed by Eurogentec, Belgium). 5 mg ofepitope peptide was immobilised to 8 ml of SulfoLink coupling Gel(Pierce Biotechnology, IL) and used to affinity purify final bleed serumfrom the rabbit with the highest antibody titre. Antibody specificitywas confirmed by western blotting, performed as previously described(22), using 2 μL of a GPC5 in vitro translation reaction and 2 μL of aluciferase in vitro translation reaction as a control. A single cleanband of the correct size was obtained for GPC5. Membrane protein wasextracted from cell lines using MEM-PER extraction reagent (PierceBiotechnology, IL) ultra-filtered using YM-30 Filter (Millipore) toremove detergent and the protein concentration determined using the BCAassay kit (Pierce Biotechnology, IL).

TABLE 1 Primers and ProbesPrimers and Probes for Genomic Quantification of GPC5 GPC5 Forward5′-CCCACCCAAATCTCATCTAGAATT-3′ 300 nM GPC5 Probe-5′-CCGGGTTCCTCCCTTTGCACATG-3′ 100 nM FAM Labelled GPC5 Reverse5′-ACGCATTGCCCAGTTGTTAGA-3′ 300 nM GJB2 Forward5′-TGGTTGCATTTAAGGTCAGAATCTT-3′  50 nM GJB2 Probe-5′-CTAGCGACTGAGCCTTGACAGCTGAGC-3′ 100 nM Vic Labelled GJB2 Reverse5′-GCAGAGGCACGTTCAGGAA-3′ 300 nMPrimers and Probes for Expression Quantification of GPC5 GPC5 Forward 5′-GGGCTGCCGGATTCG-3′ 300 nM GPC5 Probe- 5′-CGCGGGCAGGACCTGATCTTCA-3′100 nM FAM Labelled GPC5 Reverse 5′-CTGGTGCAACATGTAGGCTTTT-3′ 300 nMGAPDH PDAR Applied Biosystems Part No. 4310884E lXPrimers and Probes for Genomic Quantification of GPC6 GPC6 Forward5′-TGACCAGCTCAAGCCATTTG-3′  50 nM GPC6 Probe-5′-AGACGTGCCCCGGAAACTGAAGATTC-3′ 100 nM FAM Labelled GPC6 Reverse5′-TGAAGGCGCGGGTAACC-3′ 300 nMPrimers and Probes for Expression Quantification of GPC6 GPC6 Forward5′-AACGAGGAGGAATGCTGGAA-3′ 300 nM GPC6 Probe-5′-CACAGCAAAGCCAGATACTTGCCTGAGATC-3′ 100 nM FAM Labelled GPC6 Reverse5′-CTGGTTGGTGAGCCCATCAT-3′  50 nMPrimers for amplification of GPC5 sequence including restriction sites and kozak sequence GPC5 Forward5′-TATAAGCTTCCACCATGGACGCACAGACCTGGCCCG-3′ 300 nM GPC5 Reverse5′-CGCGTCGACTTACCAAATCCCGGGAAGTA-3′ 300 nM

Antisense Oligonucleotides (ASOS) Targeted to GPC5 and WT1.

20 mer, 2′-O-methoxyethyl (2′-MOE) chimeric oligonucleotides consistingof a central window of eight 2′-deoxy unmodified sugar residues withflanking 2′-MOE regions and a fully thioated backbone were synthesizedby Isis Pharmaceuticals Inc., as described previously (Baker, B. F.,Lot, S. S., Condon, T. P., Cheng-Flournoy, S., Lesnik, E. A., Sasmor, H.M., and Bennett, C. F. (1997) J Biol Chem 272, 11994-12000). Twentyantisense oligonucleotides targeting predicted accessible GPC5 mRNAsequences over the full length mRNA product were provided and screenedfor activity in K562 cells. ISIS 15770, sequence5′-ATGCATTCTGCCCCCAAGGA-3′, a 5-10-5 gapmer targeting murine c-rafkinase was used as a control in this screen. The two active compoundsidentified were ISIS 276107 sequence 5′-CAGCCCCCTGACAGCTCCCA-3′, andISIS 276119 sequence 5′-CCATCTGCAGCAGCTAATTC-3′. Also used as a controlwas ISIS 276124, sequence 5′-TGGATTTGCTTTACATCACT-3′.

The previously identified WT1 ASOs were ISIS 16609, sequence5′-GCCCTTCTGTCCATTTCACT-3′, targeting WT1 exon 5 (ASWT1exon 5) and ISIS16601, sequence 5′-CACATACACATGCCCTGGCC-3′, targeting the 3′-UTR regionof WT1 (ASWT13′UTR). The control ASO was ISIS 105730, sequence5′-CCATCGACCTGCACCGATCA-3′, a scrambled sequence of ASWT13′UTR,(ASWT1scram).

Assessment of GPC5 and WT1 Antisense Activity.

Antisense or control oligonucleotides were dissolved in PBS andintroduced into K562 cells by low voltage electroporation: 40 μl ofappropriately diluted ASOs were combined with 360 μl of cell suspensionat 2×10⁷ cells/ml and cells were electroporated (Bio-Rad Gene Pulser® IIElectroporation system with Pulse Controller Plus capacitance extenderaccessory module, Bio-Rad Laboratories Ltd, Hemel Hempstead, Herts, UK)using 300V and a capacitor value of 1000 μF, and diluted to 10 ml withcomplete medium. From each sample appropriate duplicate aliquots ofcells were serially diluted in complete medium for assessment ofcytotoxicity while the remaining cells were incubated at 37° C. for 24hours and RNA extracted for quantification of GPC5 and/or WT1 expressionlevels.

Clonogenic Cell Survival Assay.

In this assay, cells are grown at low density in suspension in soft agarfollowing treatment. Each colony formed derives from a single survivingcell. 2 ml aliquots of diluted cells were added to polystyrene tubes(Elkay Products (UK) Ltd., Basingstoke, Hampshire, UK) containing 3 mlof medium supplemented with 20% FCS and 0.2% Agar Noble (DIFCOLaboratories, Detroit, Mich.), incubated at 37° C., and coloniescontaining at least 32 cells counted after 14 days. The number ofcolonies formed following drug treatment were compared to the numberobtained following sham electroporation or a control scrambledoligonucleotide and expressed as % control treatment. In a number ofseparate experiments, control plating efficiencies ranged from 22-380,with 800-1600 cells typically plated. In addition, cells from thedilution series (approx 10³-10⁴ cells/ml) were allowed to grow up insuspension culture in parallel for 3-5 days in order to confirmcytotoxicity.

Production of GPC5 Construct and Transfection

Image clone 5744533 containing the full coding region of human GPC5 wasobtained from the ATCC (American Type Culture Collection at LGCPromochem, UK). The coding region was amplified using the Xpand Highfidelity PCR kit (Roche, Switzerland) and primers to produce a productcontaining an in frame kozak sequence at the 5′ end (see Table 1). Thisproduct was TA cloned into the vector pCR4-TOPO (Invitrogen, CA) andsequence confirmed using Applied Biosystems Big Dye Sequencing kitVersion 1 and a 377 ABI Prism sequencing machine. The insert wasrestriction digested with EcoR1 and re-ligated into an EcoR1 digestedvector pCMV-TnT (Promega, UK) using T4 DNA ligase (Invitrogen, CA).Purified pCMV-TnT-GPC5 and a control T7-luciferase plasmid were in vitrotranslated using Promega's Quick coupled TnT T7 in vitro translationsystem including biotin labelled lysine (Promega, UK) as permanufacturers instructions. Products were separated by SDS-PAGE, blottedonto Immoblion-P PVDF membrane (Millipore, UK) and colour developedusing a streptavidin-Alkaline phosphatase and western blue colormetricsubstrate (Promega, UK) as per manufacturers instructions.

Following cleanup of plasmids using Wizard Purefection kit (Promega, UK)plasmids were transfected into the RMS cell line T91-95 at ˜75%confluence using FuGene6 transfection reagent (Roche, Switzerland) in aratio of 3 μl FuGene:1 μg DNA. pCMV-TnT-GPC5 was co-transfected in amolar ratio of 10:1 with pTK-Hyg (Clontech, CA). A control transfectionusing empty pCMV-TnT vector in a 10:1 molar ratio with pTK-Hyg was alsoperformed. Cells were grown in Dulbecco's Modified Eagle Media (DMEM)and 10% foetal calf serum. 200 μg per ml of hygromycin (Clontech, CA)was added 48 hr post transfection in order to produce stable colonies.After two weeks healthy stable colonies were selected and expanded.Overexpression of GPC5 was confirmed by TaqMan RT-PCR using DNasetreated cDNA (DNA Free, Arabian, Tex.) and increased protein wasconfirmed by western blot analysis.

Cell Proliferation Assay

In order to assess the growth characteristics of stably transfectedcells a method based on the ability of the metabolic enzymehexosaminidase to produce a coloured product by breaking downp-Nitrophenyl-N-acetyl-β-D-glucosaminide was used as previouslydescribed to measure cell numbers (23).

Results Comparison of CGH and CESH Data

Meta-analysis of all the CGH data available in the literature showedthat the region 13q31-32 was gained in 21/87 (25%) of ARMS and 9/40(22%) of ERMS. 5/87 (6%) of the ARMS were defined as amplifying thisregion (4-10) (unpublished data). CESH analysis which detects grossdifferential expression on a region by region basis showed that 7/27(26%) of ARMS and 4/19 (21%) of ERMS showed overexpression from 13q31-32relative to normal muscle. Where CGH and CESH data was available fromthe same sample the majority of samples with gain at 13q31-32 alsoshowed overexpression from the region 13q31-32 7/8 (88%). In addition, 3samples showed overexpression from the region without any apparent gainby CGH.

Interphase FISH analysis of 13q31-32 Amplicon

Using BACs mapped to 13q31-32 it was possible to place the region ofamplification in two primary RMS samples and the cell line K562 to aregion of 13q31-32 approximately 3.8 Mb in length from ˜88,200K to˜91,900K on the physical map (Build 32) of chromosome 13 (see FIG. 1).2Mb of this amplified region had the highest copy number in both RMSsamples and this 2 Mb interval contains the gene GPC5 (Glypican 5). At˜2 Mb in length GPC5 is the second largest gene in the human genome todate (24). Amplification levels in K562 appeared lower and stopped shortof GPC6 the next annotated gene telomeric of GPC5. In addition to GPC5,the only other curated genes within this amplified region arepseudogenes (www.ncbi.nlm.nih.gov/locuslink) and the recently-identifiedC13ORF25 (33). The FISH mapping of the 13q31-q32 amplification in 5lymphoma samples by Yu et al defined a minimal amplification whichspanned the same region but extended an extra 1 Mb centromeric of GPC5(13).

Quantification of GPC5/GPC6 Genomic Copy Number and Expression

Genomic copy number of GPC5 was measured in primary tumour samples takenfrom 102 individuals with a diagnosis of RMS, of which 45 were ARMS, 51were ERMS and 5 were RMS—Not Otherwise Specified (RMS-NOS). 13 out of102 RMS (13%) show gain of GPC5 copies>1.5 times relative to normal DNA;by subtype 7/45 ARMS (16%), 6/51 ERMS (12%). The largest amplificationshowed ˜90 times more genomic copies than normal genomic DNA. This datawas concurrent with CGH and FISH data for samples where data wasavailable (4). There was no significant difference between genomic copynumber in ARMS and ERMS. All samples with gain of GPC5 copies weremeasured for their GPC6 copy number and showed no gain of genomic copynumber.

Expression of GPC5 was measured in 85 individuals of which 42 werediagnosed ARMS, 39 were diagnosed ERMS and 4 were diagnosed RMS-NOS.Expression of GPC5 is consistently greater than normal muscle and spansseveral orders of magnitude; median=83.5 times greater than normalmuscle (see FIG. 2A). Although expression in samples with GPC5amplification is always in the top quartile, overexpression is alsoapparent in some samples without a GPC5 amplification. Consequentlythere is no significant correlation between copy number and expression.Furthermore, there is no significant difference in GPC5 expressionbetween ERMS and ARMS: ARMS median=80.5 times greater than relative tonormal muscle, ERMS median=126 times greater than normal muscle. GPC6expression was virtually undetectable in all but one RMS sample tested.

Expression of C13ORF25 was also determined in the same set ofrhabdomyosarcoma cDNA samples. Results are shown in FIG. 2B. Whilst amajority of samples with genomic amplification also show relatively highexpression of C13ORF25, crucially there are 2 samples which show genomicgain but no expression. This is not the case with GPC5. Furthermore,roughly half of the samples do not show detectable expression ofC13ORF25, in contrast to GPC5 where many of the samples show expressionlevels much higher than that seen in muscle. Whilst these data suggestthat expression of C13ORF25A is affected by genomic gain, its impact onrhabdomyosarcomagenesis (if any) is likely to be less significant thanthat of GPC5.

Clinico-Pathological Correlations

Genomic gain of GPC5 copies appears to occur primarily in youngerchildren. Of the 83 samples with associated age data aged<21, all 13amplified samples were aged between 0-10 years old whereas 42 of thenon-amplified samples were aged 0-10 years and the remaining 28 wereaged 10-21. There is significant heterogeneity in the age at diagnosisof patients with gain of genomic copies of GPC5 and those without gainof genomic copies of tumours: Likelihood Ratio=8.332 n=83 p=0.0038.There is no significant difference observed between GPC5 expression orGPC5 amplification and grade, stage, time to death or event freesurvival.

GPC5 Downregulation and Cell Proliferation.

Twenty GPC5 targeted antisense oligonucleotides were screened for theability to significantly reduce both GPC5 levels and cell survival. Twoactive compounds, ISIS 276107 and ISIS 276119, were identified in thisprimary screen, reducing GPC5 levels to less than 30% control levels andreducing cell survival by greater than 80% (FIG. 3A). These results arecompared to two other typical ASOs from this screen where both GPC5levels and cell survival were unaffected. Although preliminary, thesedata suggest a correlation between downregulation of GPC5 expression andloss of cell viability. FIGS. 3B and C show the effects of ISIS 276107and a control compound (ISIS 276124) at varying concentrations in thesame assay. Panel B shows the percentage of colonies in a soft agarclonogenic assay compared to sham-treated levels following treatmentwith the active compound ISIS 107 and a control compound ISIS 276124,which has been found not to affect cell viability or GPC5 expression(data not shown). HL-60, a control leukaemic cell line which does notexpress GPC5, shows no decrease in colony number following treatmentwith either the active ISIS 107 or the control ISIS 124 compound. PanelC shows the percentage expression of GPC5 relative to sham-treatedlevels 24 hours after treatment.

GPC5 Expression in Prostate and Breast Cancer

For prostate samples, the assay was carried out as for therhabdomyosarcoma samples described above, except that expression wasmeasured relative to normal prostate cDNA synthesised from acommercially available normal prostate RNA pool as opposed to normalmuscle cDNA.

FIG. 4A shows the level of expression of GPC5 in a number of individualsamples. Several samples show greater expression than normal prostate.Furthermore, there is a significantly greater expression in prostatecancer compared with Benign Prostatic Hyperplasia (BPH) Mann-WhitneyU=16 p=0.011N=24. However the significance of results should beinterpreted with caution as there are only six BPH samples. Takentogether this data does suggest a potential role for GPC5 overexpressionin the development of prostate cancer.

FIG. 4B shows overall GPC5 expression in a larger sample of prostateadenocarcinoma (n=45) and benign prostate hyperplasia (n=25) relative tonormal prostate tissue. The samples used here include those for whichresults are shown in FIG. 4A.

For the breast cancer samples shown in FIG. 5A, measurement wasperformed relative to normal breast cDNA biopsy tissue as opposed tonormal muscle cDNA, and was performed in duplicate instead oftriplicate.

The data shows relative overexpression of GPC5 in some samples. Thehighest value was in biopsies taken from lymph nodes with metastasis.This suggests potential involvement of GPC5 in tumourigenesis in thesesamples.

Expression of GPC5 was then measured by TaqMan analysis in seven normalbreast samples and 44 breast cancer samples. The mean normal breast GPC5expression and 95% confidence intervals were calculated. Any tumoursample in which GPC5 expression exceeded the upper confidence intervalfor normal samples was said to overexpress GPC5. The samples weregrouped according to the stage of the disease (stage 1, 2 or 3). 5 outof the 6 samples which show overexpression are stage 3. There is asignificant difference in the stage of those samples which overexpressGPC5; Fisher's Exact Test p=0.017 n=44 (FIG. 5B).

Correlation with MYCN Expression

For some of the samples we already had information about expression ofMYCN from a previous TaqMan study (data not shown). It was found that inERMS and ARMS with a confirmed PAX/FOXO1A translocation that expressionof GPC5 correlated significantly with expression of MYCN:Spearman'sRho=0.497 n=38 p=0.002 and Spearman's Rho=0.399 n=26 p=0.043respectively.

Regulation of GPC5 Expression by the Wilms' Tumour Gene (WT1) Product.

Previous gene profiling studies following treatment of K562 cells withWT1 directed ASOs, identified GPC5 as a putative WT1 target gene(manuscript in preparation). Downregulation of GPC5 expression followingWT1 antisense treatment and correlating with reduced WT1 expression wasconfirmed in independent experiments (see FIG. 6). Similarly,preliminary in vivo studies have demonstrated downregulation of GPC5 inPC3M prostate cancer cells grown as subcutaneous implants in athymicnude mice treated with WT1 antisense: A 50% decrease in WT1 expressionlevels in ASWT1exon5 mice was reflected by a 30% reduction in GPC5expression (data not shown). Direct transcriptional regulation of GPC5by WT1 is likely since GPC5 has two WT1 consensus binding sites in itspromoter region.

Using the same in vivo model system we have obtained preliminaryevidence of upregulation of both WT1 and GPC5 expression in PC3M tumoursfollowing treatment of the mice with docetaxol (15 mg/kg): A nine foldincrease in WT1 expression was reflected by a 5 fold increase in GPC5expression. These studies raise the possibility that GPC5 overexpressionmay be induced by cancer chemotherapy. The PC3M tumour model isrelatively resistant to cytotoxic drug treatment giving rise to thespeculation that upregulation of GPC5 expression may contribute to poorresponse to therapy.

GPC5 Overexpression and Cell Proliferation

In order to test if overexpression of GPC5 could confer oncogenicproperties, a GPC5 construct was made under the control of a CMV(Cyto-Megalo Virus) promoter in order to constitutively express GPC5.This construct was tested by in vitro translation and was shown toproduce the appropriate size protein (63K Daltons) (see FIG. 8C). RMScell line T91-95 was stably transfected and healthy colonies werepicked. T91-95 is an RMS cell line which expresses both GPC5 and MYCN atlevels similar to that of normal muscle. 5 GPC5 transfected and 5 emptyvector control transfected colonies were randomly selected and subjectedto a cell proliferation assay.

Cells were plated at 7500 cells per 24 well plate in triplicate repeats.In order to allow cells to recover and to normalise for potentialvariations in plating efficiency and cell counting errors the firstmeasurement was taken at 16 hrs. The proliferation assay uses acolorimetric PNNAG assay in which absorbance is proportional to numberof cells. Log Normalised Growth is calculated as the mean naturallogarithm of absorbance of triplicates at 64 hours minus the meannatural logarithm of absorbance of triplicates at 16 hours. There is asignificant difference between the Log Normalised Growth in GPC5overexpressing colonies compared to control colonies p=0.027 t=2.70 n=10(FIG. 8A).

FIG. 8B shows the expression level of GPC5 as measured by TaqMan assayin GPC5 overexpressing colonies and control colonies, as compared tonormal muscle tissue.

Discussion

Our data support and characterize a potential role for the GPC5 gene, acell surface heparan sulfate proteoglycan, in the development of RMS.Analysis of previous data indicated differential overexpressioncorresponding to the 13q31-32 region, which harbors the GPC5 gene, wasfound in cases which amplify the region and also in some cases withoutamplification of both the alveolar and embryonal subtypes. This data wasmirrored by the quantitative analysis of GPC5 copy number and expressionand together suggests that overexpression of GPC5 is important in thepathogenesis of RMS of both subtypes. The data is also consistent withgene expression being up-regulated by mechanisms other than genomicamplification.

The Glypicans are a family of conserved cell surface heparan sulfateproteoglycans which are believed to modulate the activity of heparanbinding growth factors such as FGFs (fibroblast growth factors) and Wnts(25). Deregulation of other members of the glypican family has beenimplicated in development of a number of tumors. GPC1 (Glypican-1) hasbeen shown to be overexpressed in human pancreatic cancer and topositively regulate the action of HB-EGF (Heparan Binding EpidermalGrowth Factor) and FGF2 (Fibroblast Growth Factor 2) (26). Furthermore,stable transfection of a GPC1 antisense construct in the GPC1overexpressing pancreatic cancer cell line PANC-1 decreasedtumourgenicity (27). GPC1 is similarly overexpressed in breast cancerand modulates the activity of a number of heparan binding growth factors(28). GPC3 has been shown to aberrantly overexpress in 7/10neuroblastoma cell lines, 4/4 primary neuroblastoma samples and 7/7primary Wilms tumour samples (29). Expression in these tumours is shownto correlate with IGF2 expression (Insulin-like Growth Factor II) a genewhich is frequently overexpressed in RMS (30).

Examination of the only two annotated genes in the 13q31 region hasdemonstrated that the 13q31 amplification in RMS includes the gene GPC5but not GPC6. Although no other sequences from the region have beenshown to be expressed, it remains possible that as yet uncharacterisedgenes exert an effect. In contrast to Yu et al our amplification appearsto peak centrally at the gene GPC5. As we have demonstrated a functionalconsequence of elevated protein levels of GPC5 in RMS cells it is likelythat the overexpression of this gene associated with amplification inlymphoma cell lines is exerting a similar effect. In addition, we noteamplification and/or overexpression from 13q31-32 in another type ofsoft tissue sarcoma, namely leiomyosarcomas which resemble smooth muscletissue (11, 20)(Unpublished Data). Other sarcoma types have also beendocumented with amplification of this region and therefore it ispossible that up regulation of this gene is a more general feature ofsoft tissue sarcomas. This may extend to other tumour types describedwith amplification of this region such as breast cancer, small-cell lungcancer, medulloblastoma and glioblastoma (11-17). Preliminary data forthe expression levels of GPC5 in prostate and breast cancers relative tocorresponding normal tissues suggests that some of these tumoursaberrantly overexpress this gene.

Whilst amplification and/or overexpression of GPC5 are a frequentoccurrence in RMS, we provide no evidence that these are likely to be ofuse as clinical markers of prognosis. In the series studied there doesnot appear to be a significant difference in the survivalcharacteristics of patients with tumors amplifying or overexpressingGPC5 compared to those that do not. Furthermore, amplification seems topredominate in patients between 0-10 years; a clinical group withgenerally improved prognosis (31). Further analysis of a larger cohortof samples, perhaps using a tissue array, may identify sub-groups ofrhabdomyosarcoma in which deregulation of GPC5 is clinicallysignificant. Another possibility is that detection of GPC5 protein inblood serum of patients may serve as a marker of RMS or other cancers ina similar manner to which GPC1 protein in the serum identifies patientswith hepatocellular carcinoma (32).

The fact that expression of GPC5 correlates with expression of theoncogene MYCN is further evidence to support the role of GPC5 in thetumourigenesis of RMS. It is unclear from this study whether therelationship is causal. Certainly GPC5 overexpression in our in vitromodel does not cause overexpression of MYCN. It is noteworthy, however,that the sequence proximally upstream of the start of GPC5 transcriptioncontains an E-box (MYC trans-activation site) consensus sequencesuggesting potential direct trans-activation of GPC5 by MYCN.

Potentially as significant, is the apparent regulation of GPC5 by theWT1 gene product. Although not proven to be oncogenic, WT1 maycontribute to the maintenance of a malignant phenotype in leukaemias andthe large range of solid tumours where its expression is deregulated. Inaddition, WT1 has been implicated in drug resistance mechanisms and isbeing investigated as a potential target for cancer therapy. Our studiesso far indicate that GPC5 may well mediate at least some of thedownstream biological effects of WT1 expression making GPC5,potentially, the more effective target for therapeutic intervention.

In conclusion, we have established that GPC5 is amplified and/oroverexpressed in RMS and that GPC5 is a novel oncogene. GPC5 isparticularly attractive target for novel therapies for a number ofreasons. First, because it is a cell surface protein it is physicallyaccessible to a number of potential anti-GPC5 therapies. Second, becauseit potentially acts as a modulator of multiple growth factors therapieswhich reduce the function of GPC5 could therefore affect multipletumorigenic pathways. Third, because it is likely to be important in thetumorigenesis of a number of other cancer types.

REFERENCES

-   1. Galili, N., Davis, R. J., Fredericks, W. J., Mukhopadhyay, S.,    Rauscher, F. J., 3rd, Emanuel, B. S., Rovera, G., and Barr, F. G.    Fusion of a fork head domain gene to PAX3 in the solid tumour    alveolar rhabdomyosarcoma. Nat Genet, 5: 230-235, 1993.-   2. Shapiro, D. N., Sublett, J. E., Li, B., Downing, J. R., and    Naeve, C. W. Fusion of PAX3 to a member of the forkhead family of    transcription factors in human alveolar rhabdomyosarcoma. Cancer    Res, 53: 5108-5112, 1993.-   3. Davis, R. J., D'Cruz, C. M., Lovell, M. A., Biegel, J. A., and    Barr, F. G. Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14)    translocation in alveolar rhabdomyosarcoma. Cancer Res, 54:    2869-2872, 1994.-   4. Gordon, A. T., Brinkschmidt, C., Anderson, J., Coleman, N.,    Dockhorn-Dworniczak, B., Pritchard-Jones, K., and Shipley, J. A    novel and consistent amplicon at 13q31 associated with alveolar    rhabdomyosarcoma. Genes Chromosomes. Cancer, 28: 220-226, 2000.-   5. Bridge, J. A., Liu, J., Qualman, S. J., Suijkerbuijk, R., Wenger,    G., Zhang, J., Wan, X., Baker, K. S., Sorensen, P., and Barr, F. G.    Genomic gains and losses are similar in genetic and histologic    subsets of rhabdomyosarcoma, whereas amplification predominates in    embryonal with anaplasia and alveolar subtypes. Genes Chromosomes    Cancer, 33: 310-321, 2002.-   6. Menghi-Sartorio, S., Mandahl, N., Mertens, F., Picci, P., and    Knuutila, S. DNA copy number amplifications in sarcomas with    homogeneously staining regions and double minutes. Cytometry, 46:    79-84, 2001.-   7. Pandita, A., Zielenska, M., Thorner, P., Bayani, J., Godbout, R.,    Greenberg, M., and Squire, J. A. Application of comparative genomic    hybridization, spectral karyotyping, and microarray analysis in the    identification of subtype-specific patterns of genomic changes in    rhabdomyosarcoma. Neoplasia, 1: 262-275, 1999.-   8. Bridge, J. A., Liu, J., Weibolt, V., Baker, K. S., Perry, D.,    Kruger, R., Qualman, S., Barr, F., Sorensen, P., Triche, T., and    Suijkerbuijk, R. Novel genomic imbalances in embryonal    rhabdomyosarcoma revealed by comparative genomic hybridization and    fluorescence in situ hybridization: an intergroup rhabdomyosarcoma    study. Genes Chromosomes. Cancer, 27: 337-344, 2000.-   9. Weber-Hall, S., Anderson, J., McManus, A., Abe, S., Nojima, T.,    Pinkerton, R., Pritchard-Jones, K., and Shipley, J. Gains, losses,    and amplification of genomic material in rhabdomyosarcoma analyzed    by comparative genomic hybridization. Cancer Res, 56: 3220-3224,    1996.-   10. Roberts, I., Gordon, A., Wang, R., Pritchard-Jones, K., Shipley,    J., and Coleman, N. Molecular cytogenetic analysis consistently    identifies translocations involving chromosomes 1, 2 and 15 in five    embryonal rhabdomyosarcoma cell lines and a PAX-FOXO1A fusion gene    negative alveolar rhabdomyosarcoma cell line. Cytogenet Cell Genet,    95: 134-142, 2001.-   11. Wang, R., Titley, J. C., Lu, Y. J., Summersgill, B. M.,    Bridge, J. A., Fisher, C., and Shipley, J. Loss of 13q14-q21 and    gain of 5p14-pter in the progression of leiomyosarcoma. Mod Pathol,    16: 778-785, 2003.-   12. Larramendy, M. L., Tarkkanen, M., Blomqvist, C., Virolainen, M.,    Wiklund, T., Asko-Seljavaara, S., Elomaa, I., and Knuutila, S.    Comparative genomic hybridization of malignant fibrous histiocytoma    reveals a novel prognostic marker. Am J Pathol, 151: 1153-1161,    1997.-   13. Yu, W., Inoue, J., Imoto, I., Matsuo, Y., Karpas, A., and    Inazawa, J. GPC5 is a possible target for the 13q31-q32    amplification detected in lymphoma cell lines. J Hum Genet, 48:    331-335, 2003.-   14. Ojopi, E. P., Rogatto, S. R., Caldeira, J. R., Barbieri-Neto,    J., and Squire, J. A. Comparative genomic hybridization detects    novel amplifications in fibroadenomas of the breast. Genes    Chromosomes Cancer, 30: 25-31, 2001.-   15. Schmidt, H., Wurl, P., Taubert, H., Meye, A., Bache, M.,    Holzhausen, H. J., and Hinze, R. Genomic imbalances of 7p and 17q in    malignant peripheral nerve sheath tumors are clinically relevant.    Genes Chromosomes Cancer, 25: 205-211, 1999.-   16. Weber, R. G., Sabel, M., Reifenberger, J., Sommer, C.,    Oberstrass, J., Reifenberger, G., Kiessling, M., and Cremer, T.    Characterization of genomic alterations associated with glioma    progression by comparative genomic hybridization. Oncogene, 13:    983-994, 1996.-   17. Reardon, D. A., Jenkins, J. J., Sublett, J. E., Burger, P. C.,    and Kun, L. K. Multiple genomic alterations including N-myc    amplification in a primary large cell medulloblastoma. Pediatr    Neurosurg, 32: 187-191, 2000.-   18. Naumann, S., Reutzel, D., Speicher, M., and Decker, H. J.    Complete karyotype characterization of the K562 cell line by    combined application of G-banding, multiplex-fluorescence in situ    hybridization, fluorescence in situ hybridization, and comparative    genomic hybridization. Leuk Res, 25: 313-322, 2001.-   19. Lu, Y. J., Williamson, D., Clark, J., Wang, R., Tiffin, N.,    Skelton, L., Gordon, T., Williams, R., Allan, B., Jackman, A.,    Cooper, C., Pritchard-Jones, K., and Shipley, J. Comparative    expressed sequence hybridization to chromosomes for tumor    classification and identification of genomic regions of differential    gene expression. Proc Natl Acad Sci USA, 98: 9197-9202, 2001.-   20. Lu, Y. J., Williamson, D., Wang, R., Summersgill, B., Rodriguez,    S., Rogers, S., Pritchard-Jones, K., Campbell, C., and Shipley, J.    Expression profiling targeting chromosomes for tumor classification    and prediction of clinical behavior. Genes Chromosomes Cancer, 38:    207-214, 2003.-   21. Smedley, D., Hamoudi, R., Clark, J., Warren, W., Abdul-Rauf, M.,    Somers, G., Venter, D., Fagan, K., Cooper, C., and Shipley, J. The    t(8:13)(p11;q11-12) rearrangement associated with an atypical    myeloproliferative disorder fuses the fibroblast growth factor    receptor 1 gene to a novel gene RAMP. Hum Mol Genet, 7: 637-642,    1998.-   22. Perani, M., Ingram, C. J., Cooper, C. S., Garrett, M. D., and    Goodwin, G. H. Conserved SNH domain of the proto-oncoprotein SYT    interacts with components of the human chromatin remodelling    complexes, while the QPGY repeat domain forms homo-oligomers.    Oncogene, 22: 8156-8167, 2003.-   23. Landegren, U. Measurement of cell numbers by means of the    endogenous enzyme hexosaminidase. Applications to detection of    lymphokines and cell surface antigens. J Immunol Methods, 67:    379-388, 1984.-   24. Veugelers, M., De Cat, B., Delande, N., Esselens, C., Bonk, I.,    Vermeesch, J., Marynen, P., Fryns, J. P., and David, G. A 4-Mb    BAC/PAC contig and complete genomic structure of the GPC5/GPC6 gene    cluster on chromosome 13q32. Matrix Biol, 20: 375-385, 2001.-   25. Filmus, J. Glypicans in growth control and cancer. Glycobiology,    11: 19R-23R, 2001.-   26. Kleeff, J., Ishiwata, T., Kumbasar, A., Friess, H., Buchler, M.    W., Lander, A. D., and Korc, M. The cell-surface heparan sulfate    proteoglycan glypican-1 regulates growth factor action in pancreatic    carcinoma cells and is overexpressed in human pancreatic cancer. J    Clin Invest, 102: 1662-1673, 1998.-   27. Kleeff, J., Wildi, S., Kumbasar, A., Friess, H., Lander, A. D.,    and Korc, M. Stable transfection of a glypican-1 antisense construct    decreases tumorigenicity in PANC-1 pancreatic carcinoma cells.    Pancreas, 19: 281-288, 1999.-   28. Matsuda, K., Maruyama, H., Guo, F., Kleeff, J., Itakura, J.,    Matsumoto, Y., Lander, A. D., and Korc, M. Glypican-1 is    overexpressed in human breast cancer and modulates the mitogenic    effects of multiple heparin-binding growth factors in breast cancer    cells. Cancer Res, 61: 5562-5569, 2001-   29. Saikali, Z. and Sinnett, D. Expression of glypican 3 (GPC3) in    embryonal tumors. Int J Cancer, 89: 418-422, 2000.-   30. Khan, J., Wei, J. S., Ringner, M., Seal, L. H., Ladanyi, M.,    Westermann, F., Berthold, F., Schwab, M., Antonescu, C. R.,    Peterson, C., and Meltzer, P. S. Classification and diagnostic    prediction of cancers using gene expression profiling and artificial    neural networks. Nat Med, 7: 673-679, 2001.-   31. Crist, W. M., Anderson, J. R., Meza, J. L., Fryer, C., Raney, R.    B., Ruymann, F. B., Breneman, J., Qualman, S. J., Wiener, E.,    Wharam, M., Lobe, T., Webber, B., Maurer, H. M., and    Donaldson, S. S. Intergroup rhabdomyosarcoma study-IV: results for    patients with nonmetastatic disease. J. Clin. Oncol., 19: 3091-3102,    2001.-   32. Capurro, M., Wanless, I. R., Sherman, M., Deboer, G., Shi, W.,    Miyoshi, E., and Filmus, J. Glypican-3: a novel serum and    histochemical marker for hepatocellular carcinoma. Gastroenterology,    125: 89-97, 2003.-   33. Ota, A. et al. Identification and Characterization of a Novel    Gene, C13orf25, as a Target for 13q31-q32 Amplification in Malignant    Lymphoma. Cancer Res., 64: 3087-3095, 2004.

The disclosure of all references cited herein, insofar as it may be usedby those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

1-41. (canceled)
 42. A method of inhibiting proliferation of a targetcell, comprising contacting the cell with a GPC5 antagonist or a GPC5binding agent.
 43. A method according to claim 42 wherein the targetcell inappropriately expresses or overexpresses GPC5.
 44. A methodaccording to claim 42 wherein the cell inappropriately expresses oroverexpresses WT1.
 45. A method according to claim 42 wherein the cellis a cancer cell.
 46. A method according to claim 45 wherein the canceris selected from the group consisting of rhabdomyosarcoma, lymphoma,non-small cell lung cancer, bladder cancer, breast cancer, prostatecancer, a neuroglial tumour, squamous cell carcinoma of the head andneck, leukemia, leiomyosarcoma, liposarcoma, malignant fibroushistocytoma of bone or soft tissues, melanoma, mesothelioma, thyroidcancer, lung cancer, testicular cancer and ovarian cancer.
 47. A methodaccording to claim 42 wherein the cell does not carry a chromosomalamplicon at 13q31.
 48. A method according to claim 42 wherein thebinding agent binds to GPC5 protein.
 49. A method according to claim 48wherein the binding agent is an antibody or a peptide.
 50. A methodaccording to claim 48 further comprising contacting the cell with atherapeutic agent.
 51. A method according to claim 50 wherein thetherapeutic agent is associated with the binding agent.
 52. A methodaccording to claim 50 wherein the therapeutic agent is capable ofbinding to the binding agent.
 53. A method according to claim 50 whereinthe therapeutic agent comprises a moiety selected from the groupconsisting of a cytotoxic molecule, a precursor molecule capable ofbeing converted into a cytotoxic molecule by enzyme action, a cell ormolecule of the immune system, and a viral vector.
 54. A methodaccording to claim 42 wherein the binding agent binds to heparansulphate chains associated with GPC5 protein.
 55. A method according toclaim 54 wherein the binding agent is an antibody or a peptide.
 56. Amethod according to claim 42 wherein the GPC5 antagonist inhibitsactivity of GPC5 protein.
 57. A method according to claim 56 wherein theGPC5 antagonist is an antibody or a peptide.
 58. A method according toclaim 56 further comprising contacting the cell with a therapeuticagent.
 59. A method according to claim 58 wherein the GPC5 antagonistincreases the sensitivity of the cell to the therapeutic agent.
 60. Amethod of determining the susceptibility of a cancer to treatment with aGPC5 antagonist or binding agent, comprising determining the presence,absence or level of expression of GPC5 and/or WT1 in a cell from saidcancer.
 61. A method according to claim 19 further comprising the stepof determining the presence, absence or degree of chromosomalamplification at 13q31 in a cell from said cancer.
 62. A method ofdetermining the susceptibility of a cancer to treatment with a GPC5antagonist or binding agent, comprising determining the presence,absence or degree of chromosomal amplification at 13q31.
 63. A methodaccording to claim 62 further comprising determining the presence,absence or level of expression of GPC5 and/or WT1 in a cell from saidcancer.
 64. A method according to claim 60 wherein the cancer isselected from the group consisting of rhabdomyosarcoma, lymphoma,non-small cell lung cancer, bladder cancer, breast cancer, prostatecancer, a neuroglial tumour, squamous cell carcinoma of the head andneck, leukemia, leiomyosarcoma, liposarcoma, malignant fibroushistocytoma of bone or soft tissues, melanoma, mesothelioma, thyroidcancer, lung cancer, testicular cancer and ovarian cancer.
 65. A methodof screening for the presence of a cancer in a patient, the methodcomprising determining the presence, absence or level of circulatingGPC5 in the patient.
 66. A method according to claim 65 comprisingdetermining the presence, absence or level of circulating GPC5 in asample derived from the patient.
 67. A method according to claim 66wherein the sample is selected from the group consisting of blood, serumand plasma.
 68. A method according to claim 66 wherein the methodcomprises contacting the sample with a GPC5 binding agent.
 69. A methodaccording to claim 65 wherein the cancer is selected from the groupconsisting of rhabdomyosarcoma, lymphoma, non-small cell lung cancer,bladder cancer, breast cancer, prostate cancer, a neuroglial tumour,squamous cell carcinoma of the head and neck, leukemia, leiomyosarcoma,liposarcoma, malignant fibrous histocytoma of bone or soft tissues,melanoma, mesothelioma, thyroid cancer, lung cancer, testicular cancerand ovarian cancer.
 70. A method of screening for an agent capable ofinhibiting proliferation of a target cell, comprising the steps of: (i)contacting GPC5 protein with one or more candidate substances; (ii)selecting one or more candidate substances based on their ability tobind GPC5 protein; (iii) contacting said one or more selected substanceswith a target cell; and (iv) determining the effect of said selectedsubstance(s) on proliferation of said cell.
 71. A method according toclaim 70 wherein the cell inappropriately expresses or overexpressesGPC5.
 72. A method according to claim 70 wherein the cell is a cancercell.
 73. A method according to claim 72 wherein the cancer is selectedfrom the group consisting of rhabdomyosarcoma, lymphoma, non-small celllung cancer, bladder cancer, breast cancer, prostate cancer, aneuroglial tumour, squamous cell carcinoma of the head and neck,leukemia, leiomyosarcoma, liposarcoma, malignant fibrous histocytoma ofbone or soft tissues, melanoma, mesothelioma, thyroid cancer, lungcancer, testicular cancer and ovarian cancer.
 74. A method fordetermining a prognosis for a patient with breast cancer comprisingassigning a prognosis to the patient based on the expression levels ofGPC5 in a breast tumour from that patient.
 75. A method according toclaim 74 which comprises determining the presence, absence or degree ofexpression of GPC5 in vitro using a sample containing breast cancercells from the patient.
 76. A method according to claim 75 whichcomprises contacting the sample with a GPC5 binding agent.
 77. A methodfor monitoring the success of a treatment for a cancer previously foundto express GPC5, comprising determining GPC5 expression in cells of thecancer.
 78. A method according to claim 77 comprising contacting cellsof the cancer with a GPC5 binding agent.
 79. A method according to claim78 which is performed in vitro using a sample containing cancer cellsfrom the patient.
 80. A method according to claim 77 comprisingcomparing the results obtained with results of an equivalent assayperformed for the same patient before treatment and/or at an earlierstage of treatment.
 81. A method according to claim 77 comprisingdetermining the level of expression of GPC5 within cells of the cancer,or determining the number or density of cells expressing oroverexpressing GPC5.